Novel multi-purpose bioreduction system for the large-scale production of chiral alcohols

用于大规模生产手性醇的新型多用途生物还原系统

基本信息

批准号：
13853009
负责人：
SHIMIZU Sakayu
金额：
$ 78.96万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

Chiral alcohols are useful intermediates for many kinds of pharmaceuticals and chemicals. Enzymatic asymmetric reduction of prochiral carbonyl compounds is a promising method for producing chiral alcohols. There have been many attempts to construct bioreduction systems for the industrial production of chiral alcohols. This study focuses on establishment of a novel bioreduction system using an E. coli transformant co-expressing carbonyl reductase and cofactor regeneration enzyme genes. This bioreduction system is applicable to the production of many other useful chiral alcohols, by replacing the carbonyl reductase gene with other appropriate enzyme genes for carbonyl reduction. A good library of carbonyl reduction enzymes with different substrate specificities and stereospecificities has been made. Therefore, this bioreduction system could be useful as a multi-purpose catalyst for the asymmetric reduction reactions.In addition to the carbonyl reductase reactions, this co-expression system might be applicable to the reactions requiring NAD(P)H as a cofactor, such as C=C bond reductions, reductive amination and so on. Old yellow enzyme from C. macedoniensis or S. cerevisiae was found to catalyze the asymmetric hydrogenation of C=C bond of enone compounds.The direct evolution technique, such as site-directed mutagenesis, random mutagenesis, and DNA shuffling, is an effective method for improvement or expansion of carbonyl reductase functions. Furthermore, public databases of genes encoding NAD(P)H-dependent carbonyl reductases (i.e., oxidoreductases or hypothetical oxidoreductases), determined by genome projects and so on, might also be good sources for expansion of the carbonyl reductase library. These trials might be more useful and effective in combination with traditional screening of enzymes.

手性酒精是许多药物和化学物质的有用中间体。酶促羰基化合物的酶促不对称减少是产生手性醇的有前途的方法。为了构建用于手性醇的工业生产的生物补充系统的尝试。这项研究重点是使用大肠杆菌转化的共表达羰基还原酶和辅因子再生酶基因的新生物学系统。这种生物补充系统适用于通过用其他适当的酶基因代替羰基还原酶基因来生产许多其他有用的手性醇。已经制作了一个具有不同底物特异性和立体特异性的羰基还原酶库。因此，这种生物补充系统可作为用于不对称还原反应的多用途催化剂。在羰基还原酶反应外，该共表达系统可能适用于需要NAD（P）H作为Cofactor的反应，例如C = C = C键降低降低降低，还原性的降低氨化等。发现来自马其顿梭菌或酿酒酵母的旧黄色酶可以催化Enone化合物的C = C键的不对称氢化。直接进化技术，例如位置定向的诱变，随机诱变和DNA改组，是改进或膨胀碳氧化碳氧化碳纤维重型函数的有效方法。此外，编码NAD（P）H依赖性羰基还原酶（即氧化还原酶或假设氧化还原酶）的基因的公共数据库，由基因组项目等确定的，也可能是扩展羰基还原酶库的良好来源。这些试验可能与传统筛查酶结合使用更有用。